Plasma Membrane Integrity Signaling

To understand the transcriptional response of mammalian cells to plasma membrane stress we induced partial loss of plasmid membrane integrity by chemogenetic induction of pore forming proteins (Gasderimin and MLKL) or treatment with the detergent digitonin. We monitored transcription with mRNAseq in the immediate hours after plasma membrane damage and identified conserved transcriptional programs. Overall design: To induce PM damage that could be tolerated by cells, we fused proteins of human MLKL N-terminal (1-181aa) with a tandem FK506 binding domain of FKBP (Fv) and stably transduced in NIH3T3 cells. We found that the oligomerization of MLKL1-181-2xFv by B/B homodimerizer (AP20187, binds to Fv domains, hereafter referred to as "B/B") initiated PM damage by direct MLKL pore-forming. We determined that 100 nM B/B could lead to sub-lethal membrane damage to most of the cells. Alternatively, the Gasdermin family proteins, including GSDMD, can directly form PM pores. When GSDMD N-terminal (1-276aa) was fused with hormone-binding domain (HBD*), addition of 4-Hydroxytamoxifen (4-OHT) led to direct plasma membrane damage in the absence of additional signals. Parallel with hMLKL1-181-2xFv, a limited sub-lethal GSDMD (by optimized 4-OHT concentration, such as 50 nM) induced membrane damage elicited a PM damage response. Or, we used digitonin to achieve a sub-lethal PM damage. After sub-lethal PM damage, we performed several RNAseq as below.